Fabry disease (sometimes also called Anderson-Fabry disease) is a rare X-linked disorder characterized by the absence of α-galactosidase A (α-Gal A), an enzyme required for the normal processing of glycosphingolipids in mammalian lysosomes. The loss of α-Gal A leads to accumulation of the neutral globotriaosylceramide (Gb3), also known as ceramide trihexoside (CTH), within the heart, kidney, liver, and vascular endothelial cells. Renal and cardiac diseases are the most common cause of mortality and morbidity in Fabry patients [1, 2]. Hemizygous males, homozygous females, and some heterozygous females experience progressive organ dysfunction manifesting clinically as angiokeratomas, acroparathesis, stroke, cardiomyopathies, myocardian infarction and renal failure [1]. The kidney is exceptionally susceptible to damage from Gb3 deposition with several published reports of glycosphingolipid localized to the podocytes, vascular endothelial cells, and epithelial cells of the glomerulus. Loss of podocytes by apoptosis leads to glomerulosclerosis and drastically reduced kidney function. Affected individuals vary in disease progression and severity of symptoms.
Historically, treatment options for Fabry patients were limited to symptomatic relief of renal and cardiovascular complications [3]. Attempts at more severe treatments, namely organ transplantation [4,5] and plasmapheresis [6], did not prove successful. Currently, two galactosidase drugs are available for treatment of Fabry disease via enzyme replacement therapy (ERT): agalsidase alfa (Replagal®, TKT/Shire) and agalsidase beta (Fabrazyme®, Genzyme). These protein based therapeutics are administered by (approved for) intravenous injection and deliver galactosidase activity to the lysosomes of affected organs in order to reduce the level of Gb3 accumulation. Additional approaches to ERT for treatment of lysosomal storage diseases, such as Fabry disease, are needed.
An alternative strategy to ERT is substrate reduction therapy (SRT). This works on the basis of limiting the amount of pathologic substrate (i.e. Gb3) in the patient. The pathology of Fabry disease arises as a result of the patient's reduced ability to degrade Gb3 and the resulting accumulation of the substrate, and the aim of SRT is to reduce the amount of this pathologic substance that is present.
Gb3, like the other complex glycosphingolipids is synthesised from glucosylceramide (GlcCer) in the Golgi. It has recently been shown that FAPP2, a cytosolic transfer protein, has an important role in partitioning GlcCer into different pathways for downstream synthesis of different GSLs in different cellular compartments. FAPP2 has been shown to be responsible for delivering GlcCer directly to the Trans Golgi network (TGN). In the TGN the globo- and asialo-sphingolipids, including Gb3, are synthesised from GlcCer. Other GlcCer is moved through the vesicular route to the Golgi cisternae, to make the ganglio-series of sphingolipids in the Golgi cisternae. It has further been shown that FAPP-2−/− mice have a selective decrease in Gb3 in the kidneys [7].
In view of the role of FAPP2 in the synthesis of Gb3, FAPP2 represents a target for SRT for the treatment of Fabry disease. SRT has been proposed for lysosomal storage disorders such as Gaucher disease and Niemann-Pick type-C disease, and Zavesca® (Actelion) has been approved for the treatment of mild to moderate type-1 Gaucher disease patients who cannot receive the standard treatment of ERT and for the treatment of the neurological symptoms of the disease patients of all ages with Niemann-Pick type-C disease. Inhibitors of FAPP2 that are suitable for SRT of Fabry disease are, however, not currently available.
Whilst the structure of the GLTP domain of human FAPP2 has been modelled, based on the crystal structure of the human glycolipid transfer protein (GLTP) [8], a high resolution crystal structure of the glycolipid binding portion of FAPP2 is needed in order to develop FAPP2 inhibitors suitable for SRT. In particular, it is known that FAPP2 has a different lipid transfer specificity to GLTP, with FAPP2 being unable to transfer certain glycolipids that are readily transferred by GLTP, such as negatively charged glycolipids. Further, there are differences in the structure of the two proteins, which is reflected in their different helix content and the relatively low Tm for the GLTP domain of FAPP2, which exhibits thermal unfolding with a Tm of about 41° C., compared to 53° C. for GLTP [8].
Developing inhibitors that are specific for FAPP2 is of particular interest, and understanding the structure of FAPP2 and how it may differ from other related and non-related molecules is important in this process.